Double-stranded rna oligonucleotides which inhibit tyrosinase expression

ABSTRACT

Novel double-stranded RNA oligonucleotides are useful for decreasing tyrosinase expression, have cosmetic and/or pharmaceutical applications, for example are useful skin depigmenting or anti-browning agents, and can be associated with cationic particles less than or equal to 1 μm in size, having a zeta potential of from 10 to 80 mV.

CROSS-REFERENCE TO PRIORITY APPLICATION

This application is a continuation of U.S. application Ser. No.11/524,315 filed Sep. 21, 2005 which claims priority under 35 U.S.C. §119 of FR 05/09658, filed Sep. 21, 2005, both of which are herebyexpressly incorporated by reference and assigned to the assignee hereof.

BACKGROUND OF THE INVENTION

1. Technical Field of the Invention

The present invention relates to novel double-stranded RNAoligonucleotides for decreasing tyrosinase expression, to the usethereof for cosmetic and/or pharmaceutical purposes and also to theassociation thereof with cationic particles less than or equal to 1 μmin size, with a zeta potential ranging from 10 to 80 mV.

2. Description of Background and/or Related and/or Prior Art

Tyrosinase (monophenol dihydroxyl phenylalanine: oxygen oxidoreductaseEC 1.14.18.1) is the essential enzyme involved in the mechanism of skinpigmentation. It catalyzes in particular the reaction for conversion oftyrosine to Dopa (dihydroxyphenylalanine) by virtue of its hydroxylaseactivity and the reaction for conversion of Dopa to dopaquinone byvirtue of its oxidase activity. The tyrosinase acts only when it is inthe maturation state under the action of certain biological factors.

This enzyme can also be advantageous in the treatment of pathologiessuch as melanoma (Riley et al., J. Immunother., 2001, 21, 212-220) orVogt-Koyanagi-Harada disease (Read et al., Curr. Opin. Opthalmol., 2000,11, 437-442).

The mechanism of formation of skin pigmentation, i.e., the formation ofmelanin, is particularly complex and involves schematically thefollowing main steps:

Tyrosine→Dopa→Dopaquinone→Dopachrome→Melanin

Thus, the pigmentation of human skin results from the synthesis ofmelanin by dendritic cells, melanocytes. The latter contain organellescalled melanosomes, which are the site of melanin biosynthesis. It isthe melanosomes which, after migration along the dendrites, aretransferred from the melanocytes to the keratinocytes. The keratinocytesare then transported to the surface of the skin during the epidermaldifferentiation process (Gilchrest B A, Park H Y, Eller M S, Yaar M,Mechanisms of ultraviolet light-induced pigmentation, PhotochemPhotobiol., 1996; 63: 1-10; Hearing V J, Tsukamodo K, Enzymatic controlof pigmentation in mammals, FASEB J., 1991; 5: 2902-2909).

Among melanogenesis enzymes, tyrosinase is a key enzyme which catalysesthe first two steps of melanin synthesis. Homozygous mutations fortyrosinase cause oculocutaneous albinism type I characterized by acomplete absence of melanin synthesis (Toyofuku K, Wada I, Spritz R A,Hearing V J, The molecular basis of oculocutaneous albinism type 1(OCA1): sorting failure and degradation of mutant tyrosinases results ina lack of pigmentation, Biochem J., 2001; 355: 259-269).

Since hyperpigmentation disorders result from an increase in melaninproduction, the development of novel therapeutic approaches, therationale of which is based on the inhibition of tyrosinase activity, isfound to be important.

Most of the skin-lightening compounds already known arephenols/catechols. These compounds inhibit tyrosinase but most of thesecompounds are cytotoxic with respect to melanocytes, which could causepermanent depigmentation of the skin.

It therefore appears to be advantageous, for an application in humans,to have novel tyrosinase-inhibiting compounds that are both highlyeffective and exhibit good tolerance.

Of late, the use of double-stranded RNA, dsRNA, oligonucleotides, andmore particularly of siRNA oligonucleotides (of 12 to 40 nucleotides),would make it possible to obtain a specific activity in the cosmeticfield, such as skin care or hair care, but also in the dermatologicaland pharmaceutical fields.

However, the use of siRNAs in vivo is known to present variousdifficulties.

Besides the problem of the penetration of these siRNAs in order to reachthe target cells when they are applied topically, due in particular tothe difficulty in crossing the stratum corneum, experience has shownthat the administration of siRNAs could bring about the triggering of aninterferon response reported by numerous publications (Sledz C A et al.,Activation of the interferon system by short-interfering RNA, Nat CellBiol., 2003; 9:834-9. Katalin Karikó et al., Small Interfering RNAsMediate Sequence-Independent Gene Suppression and Induce ImmuneActivation by Signaling through Toll-Like Receptor 31, J. Immunol.,2004; 172: 6545-6549. Judge A D et al., Sequence-dependent stimulationof the mammalian innate immune response by synthetic siRNA, NatBiotechnol., 2005; 23:457-62) and also the induction or the inhibitionof the expression of genes not targeted by the siRNA (Jackson et al.,Expression profiling reveals off-target gene regulation by RNAi, NatBiotechnol., 2003; 21:635-7), these two phenomena are highlyundesirable.

Published U.S. Application No. 2004/0215006 describes double-strandedand anti-sense single-stranded RNA oligonucleotides which are activeagainst tyrosinase; the examples relate only to anti-sensesingle-stranded RNA oligonucleotides. In particular, it is notdemonstrated whether the siRNAs homologous to these anti-sense RNAs areeffective.

Some authors have emphasized that double-stranded RNA oligonucleotides,such as siRNAs, and anti-sense single-stranded RNA oligonucleotides havedifferent targets and that the activity of an anti-sense RNA cannot beextrapolated to the siRNA of the same sequence (Xu et al., Effectivesmall interfering RNAs and phosphorothioate anti-sense DNAs havedifferent preferences for target sites in the luciferase mRNAs, BBRC2003; 306:712-717).

Moreover, in said 2004/0215006, the concentrations of siRNA proposed arefrom 50 and 200 nM. As indicated above, Jackson et al., have described astrong positive and negative regulation of genes not targeted by thesiRNAs used at concentrations of 100 nM.

Similarly, in WO 2005/060536, which describes siRNAs that specificallyinhibit tyrosinase, the concentration ranges proposed are very wide andcan result in compositions comprising a very large amount of siRNAs forwhich a positive and negative regulation of genes not targeted by thesiRNAs may be observed.

In the context of a cosmetic or therapeutic use, this regulation ofnon-targeted genes is not acceptable, in particular due to itsunpredictable nature.

SUMMARY OF THE INVENTION

Novel siRNAs have now been developed that specifically inhibittyrosinase expression from which siRNAs have been selected which exhibitan activity such that the siRNAs can be used at a dose of less than orequal to 1 nM.

In addition to solving the problems related to the induction ofnonspecific genes, it has now been verified that these sequences ofsiRNAs do not induce an interferon response.

For this, the expression was measured of the OAS-1 and IFIT-1(interferon-inducible tetratricopeptide repeat domain) genes known to beinduced by interferon (Stefan F. Wieland et al., Searching forInterferon-Induced Genes that inhibit Hepatitis B Virus Replication inTransgenic Mouse Hepatocytes, J. of Virology 2003, 77:1227-1236)according to the protocol described in the manual of the “BLOCK-iT™ RNAiStress Response Control Kit (Human) for monitoring interferon-mediatedstress response to double-stranded RNA in human cells” marketed byInvitrogen.

Furthermore, the association of these tyrosinase-specific siRNAs withcationic particles less than or equal to 1 μm in size, with a zetapotential of 10 to 80 mV, makes it possible to very significantlyimprove their penetration into the target cells of a three-dimensionalmodel such as the skin. Once penetrated, the tyrosinase-specific siRNAbecomes active.

The penetration can be evaluated by means of a fluorescent labelattached to the siRNA and its activity can be evaluated by quantifyingthe targeted messenger by quantitative PCR or by assaying the proteincorresponding to the targeted messenger.

The cationic particles of the invention may be surfactant micelles,micelles of block or non-block polymers, cationic liposomes andniosomes, cationic oleosomes, cationic nanoemulsions, and also cationicorganic or inorganic particles and nanocapsules.

Thus, the present invention features a double-stranded RNAoligonucleotide (also called dsRNA or siRNA) selected from among theoligonucleotides of sequence SEQ ID NOS. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47 and 48, optionally modified.

The use of dsRNA and, more particularly, of siRNA (for short interferingRNA) makes it possible to obtain an activity of specific inhibition ofthe synthesis of a target protein by degradation of the mRNA encodingthe protein. The degradation of the target mRNA is obtained through theactivation of the RISC complex (RNA Induced Silencing Complex) which hasits effect through the binding of the anti-sense strand of the dsRNA tothe mRNA (see Tuschl T. Chem. Biochem., 2001; 2:239-245; Nykanen A & al,Cell 2001; 107:309-321; Dorsett Y. and Tuschl T. Nat Rev Drug Discov.,2004; 3:318-329; Downward J. BMJ. 2004; 328:1245-1248; Shanker P. etal., JAMA 2005; 293:1367-1373).

The molecular mechanism implemented involves double-stranded RNAfragments consisting of 12 to 40 nucleotides, preferably of 23 to 27nucleotides.

These double-stranded RNA oligonucleotides can preferably be composed ofa homologous sense strand and of an anti-sense strand complementary tothe sequence of the mRNA of human tyrosinase (GenBank accession numberNM_(—)000372). The double-stranded RNA oligonucleotide has blunt ends orunpaired ends of 2 to 6 nucleotides.

The double-stranded RNA oligonucleotides can be synthesized according tonumerous manual or automatic, in vivo or in vitro synthesis methods.

The in vitro synthesis methods may be chemical or enzymatic, for exampleusing an RNA polymerase (by way of example, T3, T7 or SP6) which willcarry out the transcription of a selected DNA (or cDNA) sequence model.

DETAILED DESCRIPTION OF BEST MODE AND SPECIFIC/PREFERRED EMBODIMENTS OFTHE INVENTION

Numerous methods for the in vivo synthesis of double-stranded RNA aredescribed in the literature; they can be carried out in variousbacterial cell types or cell types from higher organisms (Sambrook etal., Molecular Cloning, A Laboratory Manual, Second Edition (1989), DNAcloning, volume I and II, D. N. Glover (ed. 1985), OligonucleotideSynthesis, M. J. Gaits (ed. 1984), Nucleic Acid Hybridation, B. D. Hamesand S. J. Higgins (ed. 1984), Transcription and Translation B. D. Hamesand S. J. Higgins (ed. 1984), Animal Cell Culture, R. I. Freshney (ed.1986), Immobilized Cells and Enzymes, IRL Press (1986), B. Pertal, APractical Guide to Molecular Cloning (1984), Gene Transfer Vectors forMammalian Cells, J. H. Miller and M. P. Calos, Cold Spring HarborLaboratory (ed. 1987), Methods in Enzymology, vol. 154, Wu and Grossman,and 155, Wu, Mayer and Walker (1987), Immunochemical Methods in Cell andMolecular Biology, Academic Press, London, Scopes (1987), ProteinPurification: Principle and Practice, 2nd ed., Springer-Verlag, N.-Y.and Handbook of Experimental Immunology, vol. I-IV, C. D. Weir and C. C.Blackwell (1986)). Reference may also be made to the synthesis methodsdescribed in WO 01/36646, WO 01/75164 and U.S. Patent PublishedApplication No. 2003/0088087.

Preferably, the double-stranded RNA oligonucleotides according to theinvention are modified through the addition of an —O-methyl group in the2′-position.

The double-stranded RNA oligonucleotides can be subjected to variousmodifications, they can in particular be modified as described inPublished U.S. Application No. 2004/014956.

Advantageously, the modifications will result in double-stranded RNAoligonucleotides which are more stable, and, more preferably,double-stranded RNA oligonucleotides which have been rendered furtivewith respect to the interferon response; this property is essential foruse in vivo. The double-stranded RNA oligonucleotides may also have asense sequence modified in such a way that it cannot be incorporatedinto the RISC and therefore cannot induce side effects.

Preferably, the double-stranded RNA oligonucleotides of SEQ ID NOS. 1,2, 4, 13, 16, 35, 37, 40 and 42, having a tyrosinase mRNA degradationefficiency of greater than 50% at 0.016 nM, measured according to theprotocol of Example 2 hereinafter, will be used.

The subject of the present invention also relates to a compositioncomprising at least one double-stranded RNA oligonucleotide selectedfrom among the oligonucleotides of sequence SEQ ID NOS. 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47 and 48.

In this composition, the double-stranded RNA oligonucleotide is presentat a concentration of less than or equal to 100 μM, preferably less thanor equal to 10 μM, and more preferably less than or equal to 1 μM.

The compositions according to the invention are preferably suitable fortopical administration to the surface of the body, i.e., the skin, theinteguments, the mucous membranes and the eyes.

In a preferred embodiment of the present invention, the double-strandedRNA oligonucleotides are associated with a cationic particle less thanor equal to 1 μm in size, with a zeta potential of from 10 to 80 mV.

In this embodiment, the double-stranded RNA oligonucleotides will adhereto the surface of the particle, the latter serving as a carrier to causeit to penetrate into the structures of the skin and into the targetcells of the skin, of the mucous membranes or else of the eyes forcosmetic, dermatological and/or ophthalmic applications.

The cationic particles according to the invention are particles that areless than or equal to 1 μm, preferably less than or equal to 500 nm,more preferably less than or equal to 300 nm, in size, it being possibleto measure said size with, for example, a laser particle sizer type BI90plus from the company Brookhaven, and that have a zeta potential of from10 to 80 mV which can be measured with a zetameter type DELSA 440 fromthe company Coultronics.

The cationic particle can be selected from surfactant micelles, inparticular micelles of nonionic amphiphilic surfactants and of cationicsurfactants, block polymer micelles, in particular micelles of acationic amphiphilic block polymer, micelles of a nonionic amphiphilicblock polymer and of a cationic amphiphilic block polymer and micellesof a nonionic amphiphilic block polymer and of a cationic surfactant,liposomes of nonionic and cationic surfactants, niosomes, oleosomes,particles of nanoemulsions, nanocapsules, organic particles or inorganicparticles.

Listed below, and in a non-exhaustive manner, are cationic particleswhich can be used according to the invention.

Surfactant Micelles:

To recall, micelles are aggregates that are spontaneously formed byamphiphilic molecules when they are solubilized in water or oil, beyonda certain concentration referred to as critical: the CMC.

The micelles which can be used in the context of the invention includeat least one cationic surfactant. This cationic surfactant can beassociated with one or more nonionic amphiphilic surfactants.

Those skilled in the art will advantageously select the nonionic andcationic surfactants from McCutcheons 1998 “emulsifiers and detergents”and from the subsequent editions.

By way of non-limiting examples, the cationic surfactants which can beused in the context of the invention are listed hereinafter.

Without this being limiting, the nonionic surfactants which can be usedare: alkyl and polyalkyl (C6 to C30, saturated or unsaturated, branchedor unbranched) esters or ethers of PEO, of glycerol and of polyglycerol,of sorbitan which may or may not be oxyethylenated, of sucrose, ofglucose which may or may not be oxyethylenated, of maltose, of PPO-PEO.

In the case of a mixture between nonionic surfactants and cationicsurfactants, the respective proportions thereof, by weight, will be from99/1 and 1/99.

The amount of surfactants forming the micelles will be dependent on theCMC of the latter. However, in the context of the invention, theconcentration of micellar surfactants will range from 0.1 to 10%, andpreferably from 0.2 to 5%, by weight relative to the total weight of thecomposition.

Block Polymer Micelles:

The micelles of amphiphilic block polymers can be prepared according tothe method described in WO 04/035013.

The block copolymers used for the preparation of the micelles associatedwith the dsRNAs according to the invention are in particular amphiphilicblock polymers which are preferably nonionic, diblock or triblock, andwhich can form micelles on contact with water. They are in particular ofdiblock (A-B) or triblock (A-B-A) type, A corresponding to a nonionichydrophilic polymeric block and B to a hydrophobic polymeric block. Themolecular weight of the polymers can be from 1000 and 100 000 and theA/B ratio can be from 1/100 and 50/1.

In the context of the invention, three types of micelles can be used:

-   -   micelles of a cationic amphiphilic block polymer;    -   mixed micelles of a nonionic amphiphilic block polymer        associated with a cationic amphiphilic block polymer; and    -   mixed micelles of a nonionic amphiphilic block polymer        associated with a cationic surfactant.

The nonionic hydrophilic polymeric block can be selected frompolyethylene oxide (PEO) and polyvinylpyrrolidone (PVP).

The hydrophobic polymeric block can be selected from polystyrene,poly(tert-butylstyrene), poly(methyl methacrylate), poly(ethylacrylate), poly(butyl acrylate), poly(butyl methacrylate), poly(vinylacetate), polycaprolactones, polycaprolactams, polydimethylsiloxanes,poly(C₃-C₆ alkylene oxide)s, poly(aspartic acid), poly(lactic acid),poly(glycolic acid), polyleucine, polybutadienes, polyethylenes,polypropylenes, polybutylenes.

The block copolymer is preferably selected from among the followingblock copolymers:

-   -   polypropylene oxide/polyethylene oxide    -   polystyrene/polyoxyethylene    -   poly(methyl methacrylate)/polyoxyethylene    -   poly(butyl methacrylate)/polyoxyethylene    -   polyoxybutylene/polyoxyethylene    -   polycaprolactone/polyoxyethylene    -   polyethylene/polyoxyethylene    -   polyoxyethylene/polyoxybutylene/polyoxyethylene.

In the context of the invention, it is necessary to add to the micellarcomposition:

a cationic amphiphilic block polymer, one of the blocks of which iscationic and can be selected, by way of example, from among:

-   -   poly(trimethylethylammonium methacrylate);    -   quaternized poly(dimethylaminoethyl methacrylate);    -   polymethylvinylimidazolium;    -   poly(vinylbenzyltrimethylammonium chloride).

The association of a nonionic amphiphilic block polymer with a cationicamphiphilic block polymer is such that the ratio between the two willrange from 99/1 to 1/99;

and/or

at least one cationic surfactant as listed hereinafter.

In this case, the respective ratio of the nonionic amphiphilic blockpolymer to the cationic surfactant will range from 50/50 to 99/1.

In the context of the invention, the concentration of micellar blockpolymers, which may or may not be associated with a cationic surfactant,will range from 0.1 to 10%, and preferably from 0.2 to 5%, by weightrelative to the total weight of the composition.

In one embodiment of the invention, it is also possible to form micellesconsisting of cationic amphiphilic block polymers as described above.

Liposomes and Niosomes:

The nonionic amphiphilic lipids capable of forming nonionic liposomesare in particular those described in EP-0-582,503.

In particular, the nonionic amphiphilic lipids can be a mixture ofesters of at least one polyol selected from the group consisting ofpolyethylene glycol having from 1 to 60 ethylene oxide units, sorbitan,sorbitan bearing 2 to 60 ethylene oxide units, glycerol bearing 2 to 30ethylene oxide units, polyglycerols containing 2 to 15 glycerol units,sucroses, glucoses bearing 2 to 30 ethylene oxide units, and of at leastone fatty acid containing a linear or branched, saturated or unsaturatedC₅-C₁₇ alkyl chain, the number of alkyl chains per polyol group rangingfrom 1 to 10.

The expression “mixture of esters” covers not only mixtures of pureesters of different chemical families, but also covers any product whichcontains several chemically pure esters of a polyol of the same familyin variable proportions. This is, in particular, the case of productshaving a statistical formula, in their hydrophilic portion, for examplea polyglyceryl ester of formula CO—(OCH₂—CHOH—CH₂)_(n)—OH where n is astatistical value and which can contain various proportions of estersfor which n=1, n=2, n=3, n=4, etc.; it is also the case of esterscontaining several alkyl chains in their lipophilic portion, such ascocoates, which contain C₅ to C₁₇ alkyl chains, or isostearates, wherethe C₁₇ alkyl chains are a complex mixture of isomeric forms; it is alsothe case of products consisting of mixtures of mono-, di-, tri- orpolyesters of the same polyol. It should be noted that a product whichcontains only a single ester capable of forming vesicles and impuritiesof another type cannot be used according to the invention.

Commercial esters which can be used alone according to the invention,because they are in reality mixtures of esters, are, for example, asfollows:

the partial esters of sorbitan (or sorbitol anhydride) and of a fattyacid, marketed under the trademarks “SPAN 20, 40, 60 and 80” by “ICI”;

the sorbitan isostearate marketed under the trademark “SI 10 R NIKKOL”by “NIKKO”;

the sorbitan stearate bearing 4 ethylene oxide units, marketed under thetrademark “TWEEN 61” by “ICI”;

the polyethylene glycol stearate containing 8 ethylene oxide units,marketed under the trademark “MYR J 45” by “ICI”;

the polyethylene glycol monostearate of formula EMI6.1, in which formulan is equal to 4, marketed under the trademark “MYS 4” by “NIKKO”;

the polyethylene glycol stearate, molecular weight 400, chemical qualityor quality produced by biotechnology, marketed by “UNICHEMA”;

the diglyceryl stearate bearing 4 ethylene oxide units, marketed underthe trademark “HOSTACERINE DGS” by “HOECHST”;

the tetraglyceryl stearate marketed under the trademark “TETRAGLYN 1S”by “NIKKO”;

the diglyceryl isostearate, marketed by “SOLVAY”;

the diglyceryl distearate marketed under the trademark “EMALEX DSG 2” by“NIHON”;

the sucrose mono-, di- and tripalmitostearates marketed under thetrademarks “F50, F70, F110 and F160 CRODESTA” by “CRODA”;

the mixture of sucrose monopalmitostearate and sucrosedipalmitostearate, marketed under the trademark “GRILLOTEN PSE 141 G” by“GRILLO”;

the mixture of sucrose stearate and sucrose cocoate, marketed under thetrademark “ARLATONE 2121” by “ICI”;

the methylglucose distearate bearing 20 ethylene oxide units, marketedunder the trademark “GLUCAM E 20 DISTEARATE” by “AMERCHOL”.

Mixtures of these products, which are already mixtures, with oneanother, or mixtures of these products with pure products can, ofcourse, be used.

The cationic surfactants are advantageously selected from the listhereinafter and such that they confer on the dispersion a pH of from 5to 8; the weight ratio of the amount of nonionic amphiphilic lipids tothe amount of cationic surfactants in the lipid phase being from 50/1 to50/25, and the weight ratio of the lipid phase to the aqueous phase of adispersion being from 1/1000 to 300/1000.

Oleosomes:

The oleosomes relating to the invention are described in EP-0-705,593.They involve an emulsion of the oil-in-water type made up of oilyglobules provided with a lamellar liquid crystal coating, dispersed inan aqueous phase, characterized in that each oily globule is, as a unit,coated with a monolamellar or oligolamellar layer (1 to 10 sheets thatcan be visualized by transmission electron microscopy aftercryofracture) obtained from at least one lipophilic surfactant, at leastone hydrophilic surfactant and at least one cationic surfactantconferring on the emulsion a pH ranging from 5 to 8, the coated oilyglobules having an average diameter of less than 500 nanometers.

The lipophilic surfactant and the hydrophilic surfactant each contain atleast one saturated fatty chain having more than approximately 12 carbonatoms. Even more preferably, this fatty chain contains from 16 to 22carbon atoms.

According to another preferred embodiment of the invention, thelipophilic surfactant has an HLB of from approximately 2 toapproximately 5. As is well known, the term HLB (Hydrophilic-LipophilicBalance) means the balance between the size and the strength of thehydrophilic group and the size and the strength of the lipophilic groupof the surfactant.

Examples of such lipophilic surfactants are sucrose distearate,diglyceryl distearate, tetraglyceryl tristearate, decaglyceryldecastearate, diglyceryl monostearate, hexaglyceryl tristearate,decaglyceryl pentastearate, sorbitan monostearate, sorbitan tristearate,diethylene glycol monostearate, the glyceryl ester of palmitic acid andstearic acid, polyoxyethylenated monostearate 2 OE (comprising 2oxyethylene units), glyceryl monobehenate and dibehenate, andpentaerythritol tetrastearate.

The hydrophilic surfactant preferably has an HLB of from approximately 8to approximately 12.

As examples of such hydrophilic surfactants, mention may be made of thefollowing compounds: polyoxyethylenated sorbitan monostearate 4 OE,polyoxyethylenated sorbitan tristearate 20 OE, polyoxyethylenatedmonostearate 8 OE, hexaglyceryl monostearate, polyoxyethylenatedmonostearate 10 OE, polyoxyethylenated distearate 12 OE andpolyoxyethylenated methylglucose distearate 20 OE.

The cationic surfactants can advantageously be selected from among thecompounds mentioned hereinafter.

Nanoemulsions:

The cationic particles can also be selected from oil-in-waternanoemulsions comprising an oily phase dispersed in an aqueous phase,the oil globules of which have a number-average size of less than 100nm, characterized in that they comprise at least one amphiphilic lipidcomprising at least one nonionic amphiphilic lipid and a cationicamphiphilic lipid, the oily phase and the amphiphilic lipids beingpresent at a content such that the oily phase/amphiphilic lipid weightratio ranges from 3 to 10.

The nanoemulsions generally have a transparent to bluish appearance. Thetransparency of said nanoemulsions is measured by means of a coefficientof transmittance at 600 nm ranging from 10 to 90%, or else by means ofturbidity. The turbidity of the compositions of the invention rangesfrom 60 to 400 NTU, and preferably from 70 to 300 NTU, which turbidityis measured using a HACH portable turbidity meter—model 2100 P atapproximately 25° C.

The oil globules of the nanoemulsions of the invention have anumber-average size of less than 100 nm, and preferably ranging from 20to 80 nm, and more preferably from 40 to 60 nm. Decreasing the size ofthe globules makes it possible to promote penetration of the activeagents into the superficial layers of the skin (carrier effect).

The nanoemulsions in accordance with the invention are preferablyprepared at temperatures ranging from 4 to 45° C. and are thuscompatible with thermosensitive active agents.

These dispersions are in particular described in the followingapplications: EP-0-728,460, EP-0-879,589, EP-1-010,413, EP-1-010,414,EP-1-010,416, EP-1-013,338, EP-1-016,453, EP-1-018,363, EP-1-025,898 andEP-1-120,102. In all of these applications, it is specified that, inorder to improve particle stability, an ionic surfactant will be addedto the nonionic surfactant (or mixture).

In the case of the present application, one or more cationic surfactantswill be exclusively used as ionic surfactant.

The proportions indicated in the references above are to be conservedand, by way of example of a cationic surfactant, the list common to allthe particles will be selected.

The nonionic surfactants, preferably water-soluble or water-dispersible,contain at least one hydrophobic block and at least one hydrophilicblock.

The nonionic amphiphilic lipids of the invention are preferably selectedfrom among:

1/silicone surfactants,

2/amphiphilic lipids which are liquid at a temperature less than orequal to 45° C., selected from esters of at least one polyol or at leastone fatty acid containing at least one linear or branched, saturated orunsaturated, and in particular unsaturated or branched, C₈-C₂₂ alkylchain, the polyol being selected from the group consisting ofpolyethylene glycol having from 1 to 60 ethylene oxide units, sorbitan,glycerol which may contain from 2 to 30 ethylene oxide units, andpolyglycerols having from 2 to 15 glycerol units,

3/esters of a fatty acid and of a sugar and ethers of a fatty alcoholand of a sugar,

4/surfactants which are solid at a temperature equal to 45° C., selectedfrom among glycerol fatty esters, sorbitan fatty esters andoxyethylenated sorbitan fatty esters, ethoxylated fatty ethers andethoxylated fatty esters,

5/block copolymers of ethylene oxide (A) and of propylene oxide (B), andmixtures of these surfactants.

1/The silicone surfactants which can be used according to the inventionare silicone compounds containing at least one oxyethylenated chain—OCH₂CH₂— and/or oxypropylenated chain —OCH₂CH₂CH₂—; exemplary are thosedescribed in U.S. Pat. Nos. 5,364,633 and 5,411,744.

Preferably, the silicone surfactant used according to the presentinvention is a compound of formula (II):

in which:

R₁, R₂ and R₃, independently of one another, represent a C₁-C₆ alkylradical or a —(CH₂)_(x)—(OCH₂CH₂)_(y)—(OCH₂CH₂CH₂)_(z)—OR₄ radical, atleast one radical R₁, R₂ or R₃ not being an alkyl radical; R₄ beinghydrogen, an alkyl radical or an acyl radical;

A is an integer ranging from 0 to 200;

B is an integer ranging from 0 to 50; with the proviso that A and B arenot equal to zero at the same time;

x is an integer ranging from 1 to 6;

y is an integer ranging from 1 to 30;

z is an integer ranging from 0 to 5.

According to another preferred embodiment of the invention, in thecompound of formula (X), the alkyl radical is a methyl radical, x is aninteger ranging from 2 to 6 and y is an integer ranging from 4 to 30.

By way of example of silicone surfactants of formula (II),representative are the compounds of formula (III):

in which A is an integer ranging from 20 to 105, B is an integer rangingfrom 2 to 10 and y is an integer ranging from 10 to 20.

By way of example of silicone surfactants of formula (II), mention mayalso be made of the compounds of formula (IV):

H—(OCH₂CH₂)_(y)—(CH₂)₃-[(CH₃)₂SiO]_(A′)—(CH₂)₃—(OCH₂CH₂)_(y)—OH  (IV)

in which A′ and y are integers ranging from 10 to 20.

As silicone surfactants, use may particularly be made of those marketedby Dow Corning under the trademarks DC 5329, DC 7439-146, DC 2-5695 andQ4-3667. The compounds DC 5329, DC 7439-146 and DC 2-5695 are compoundsof formula (XI) where, respectively, A is 22, B is 2 and y is 12; A is103, B is 10 and y is 12; A is 27, B is 3 and y is 12.

The compound Q4-3667 is a compound of formula (IV) where A is 15 and yis 13.

2/The amphiphilic lipids which are liquid at a temperature less than orequal to 45° C. can in particular be selected from among:

the polyethylene glycol isostearate of molar weight 400 (CTFA name:PEG-8 Isostearate), marketed under the trademark Prisorine 3644 byUNICHEMA;

the diglyceryl isostearate marketed by SOLVAY;

the polyglyceryl laurate containing 2 glycerol units (polyglyceryl-2laurate), marketed under the trademark Diglycerin-monolaurate by SOLVAY;

the sorbitan oleate marketed under the trademark SPAN 80 by ICI;

the sorbitan isostearate marketed under the trademark NIKKOL SI 10R byNIKKO;

the α-butylglucoside cocoate or the α-butylglucoside caprate marketed byULICE.

3/The esters of a fatty acid and of a sugar, which can be used asnonionic amphiphilic lipids in a nanoemulsion according to theinvention, are preferably solid at a temperature of less than or equalto 45° C. and can be selected in particular from among the groupcomprising esters or mixtures of esters of a C₈-C₂₂ fatty acid and ofsucrose, of maltose, of glucose or of fructose, and esters or mixturesof esters of a C₁₄-C₂₂ fatty acid and of methylglucose.

The C₈-C₂₂ or C₁₄-C₂₂ fatty acids which form the fatty unit of theesters which can be used in the nanoemulsion of the invention contain asaturated or unsaturated linear alkyl chain having, respectively, from 8to 22 or from 14 to 22 carbon atoms. The fatty unit of the esters can inparticular be selected from stearates, behenates, arachidonates,palmitates, myristates, laurates, caprates and mixtures thereof.Stearates are preferably used.

By way of example of esters or of mixtures of esters of a fatty acid andof sucrose, of maltose, of glucose or of fructose, representative aresucrose monostearate, sucrose distearate, sucrose tristearate andmixtures thereof, such as the products marketed by Croda under thetrademark Crodesta F50, F70, F110 and F160 having, respectively, an HLB(Hydrophilic-Lipophilic Balance) of 5, 7, 11 and 16; and by way ofexample of esters or of mixtures of esters of a fatty acid and ofmethylglucose, mention may be made of polyglyceryl-3 methylglucosedistearate, marketed by Goldschmidt under the trademark Tego-care 450.Mention may also be made of monoesters of glucose or of maltose such asmethyl o-hexadecanoyl-6-D-glucoside and o-hexadecanoyl-6-D-maltoside.

The ethers of a fatty alcohol and of a sugar, which can be used asnonionic amphiphilic lipids in the nanoemulsion according to theinvention, are solid at a temperature of less than or equal to 45° C.and can be selected in particular from among the group comprising ethersor mixtures of ethers of a C₈-C₂₂ fatty alcohol and of glucose, ofmaltose, of sucrose or of fructose, and ethers or mixtures of ethers ofa C₁₄-C₂₂ fatty alcohol and of methylglucose. They are in particularalkylpolyglucosides.

The C₈-C₂₂ or C₁₄-C₂₂ fatty alcohols which form the fatty unit of theethers which can be used in the nanoemulsion of the invention contain asaturated or unsaturated linear alkyl chain having, respectively, from 8to 22 or from 14 to 22 carbon atoms. The fatty unit of the ethers can inparticular be selected from decyl, cetyl, behenyl, arachidyl, stearyl,palmityl, myristyl, lauryl, capryl and hexadecanoyl units, and mixturesthereof such as cetearyl.

By way of example of ethers of a fatty alcohol and of a sugar, mentionmay be made of alkylpolyglucosides such as decylglucoside andlaurylglucoside, marketed, for example, by Henkel under the respectivetrademarks Plantaren 2000 and Plantaren 1200, cetostearylglucosideoptionally as a mixture with cetostearyl alcohol, marketed, for example,under the trademark Montanov 68 by Seppic, under the trademark Tego-careCG90 by Goldschmidt and under the trademark Emulgade KE3302 by Henkel,and arachidylglucoside, for example in the form of the mixture ofarachidyl and behenyl alcohols and of arachidylglucoside, marketed underthe trademark Montanov 202 by Seppic.

As a nonionic amphiphilic lipid of this type, use is more particularlymade of sucrose monostearate, sucrose distearate, sucrose tristearateand mixtures thereof, polyglyceryl-3 methylglucose distearate andalkylpolyglucosides.

4/The glycerol fatty esters which can be used as nonionic amphiphiliclipids in the nanoemulsion according to the invention, which are solidat a temperature of less than or equal to 45° C., can be selected inparticular from the group comprising the esters formed from at least oneacid containing a saturated linear alkyl chain having from 16 to 22carbon atoms, and from 1 to 10 glycerol units. One or more of theseglycerol fatty esters can be used in the nanoemulsion of the invention.

These esters can in particular be selected from among stearates,behenates, arachidates, palmitates and mixtures thereof. Stearates andpalmitates are preferably used.

By way of example of a surfactant which can be used in the nanoemulsionof the invention, mention may be made of decaglyceryl (10 units ofglycerol) monostearate, distearate, tristearate and pentastearate (CTFAnames: Polyglyceryl-10 stearate, Polyglyceryl-10 distearate,Polyglyceryl-10 tristearate, Polyglyceryl-10 pentastearate), such as theproducts marketed under the respective trademarks Nikkol Decaglyn 1-S,2-S, 3-S and 5-S by Nikko, and diglyceryl monostearate (CTFA name:Polyglyceryl-2 stearate) such as the product marketed by Nikko under thetrademark Nikkol DGMS.

The sorbitan fatty esters which can be used as nonionic amphiphiliclipids in the nanoemulsion according to the invention, which are solidat a temperature of less than or equal to 45° C., are selected inparticular from among the group comprising esters of a C₁₆-C₂₂ fattyacid and of sorbitan and oxyethylenated esters of a C₁₆-C₂₂ fatty acidand of sorbitan. They are formed from at least one fatty acid containingat least one saturated linear alkyl chain having respectively from 16 to22 carbon atoms, and from sorbitol or ethoxylated sorbitol. Theoxyethylenated esters generally contain from 1 to 100 ethylene oxideunits, and preferably from 2 to 40 ethylene oxide (EO) units.

These esters can in particular be selected from among stearates,behenates, arachidates, palmitates, and mixtures thereof. Stearates andpalmitates are preferably used.

By way of example of a sorbitan fatty ester and of an oxyethylenatedsorbitan fatty ester, which can be used in the nanoemulsion of theinvention, representative are the sorbitan monostearate (CTFA name:Sorbitan stearate) marketed by ICI under the trademark Span 60, thesorbitan monopalmitate (CTFA name: Sorbitan palmitate) marketed by ICIunder the trademark Span 40, and the sorbitan tristearate 20 EO (CTFAname: Polysorbate 65) marketed by ICI under the trademark Tween 65.

The ethoxylated fatty ethers which are solid at a temperature of lessthan or equal to 45° C., which can be used as nonionic amphiphiliclipids in the nanoemulsion according to the invention, are preferablyethers formed from 1 to 100 ethylene oxide units and from at least onefatty alcohol chain having from 16 to 22 carbon atoms. The fatty chainof the ethers can in particular be selected from among behenyl,arachidyl, stearyl and cetyl units, and mixtures thereof such ascetearyl. By way of example of ethoxylated fatty ethers, mention may bemade of ethers of behenyl alcohol comprising 5, 10, 20 and 30 ethyleneoxide units (CTFA names: Beheneth-5, Beheneth-10, Beheneth-20,Beheneth-30), such as the products marketed under the trademarks NikkolBB5, BB10, BB20 and BB30 by Nikko, and the ether of stearyl alcoholcomprising 2 ethylene oxide units (CTFA name: Steareth-2), such as theproduct marketed under the trademark Brij 72 by ICI.

The ethoxylated fatty esters which are solid at a temperature of lessthan or equal to 45° C., which can be used as nonionic amphiphiliclipids in a nanoemulsion according to the invention, are esters formedfrom 1 to 100 ethylene oxide units and from at least one fatty acidchain having from 16 to 22 carbon atoms. The fatty chain of the esterscan in particular be selected from among stearate, behenate, arachidateand palmitate units, and mixtures thereof. By way of example ofethoxylated fatty esters, mention may be made of the ester of stearicacid comprising 40 ethylene oxide units, such as the product marketedunder the trademark Myrj 52 (CTFA name: PEG-40 stearate) by ICI and alsothe ester of behenic acid comprising 8 ethylene oxide units (CTFA name:PEG-8 behenate), such as the product marketed under the trademarkCompritol HD5 ATO by Gattefosse.

5/The block copolymers of ethylene oxide and of propylene oxide, whichcan be used as nonionic amphiphilic lipids in the nanoemulsion accordingto the invention, can be selected in particular from among the blockcopolymers of formula (V):

HO(C₂H₄O)x(C₃H₆O)y(C₂H₄O)zH  (V)

in which x, y and z are integers such that x+z ranges from 2 to 100 andy ranges from 14 to 60, and mixtures thereof, and more particularly fromthe block copolymers of formula (V) having an HLB ranging from 2 to 16.

These block copolymers can in particular be selected from amongpoloxamers, and in particular from Poloxamer 231 such as the productmarketed by ICI under the trademark Pluronic L81 of formula (V) withx=z=6, y=39 (HLB 2); Poloxamer 282 such as the product marketed by ICIunder the trademark Pluronic L92 of formula (V) with x=z=10, y=47 (HLB6); and Poloxamer 124 such as the product marketed by ICI under thetrademark Pluronic L44 of formula (V) with x=z=11, y=21 (HLB 16).

As nonionic amphiphilic lipids, representative are the mixtures ofnonionic surfactants described in EP-A-705593, incorporated herein forreference.

Among the nonionic amphiphilic lipids, use may in particular be made of:

PEG 400 isostearate or PEG-8 isostearate (containing 8 mol of ethyleneoxide),

diglyceryl isostearate,

polyglyceryl monolaurate containing 2 units of glycerol and polyglycerylstearates containing 10 units of glycerol,

sorbitan oleate,

sorbitan isostearate, and mixtures thereof.

The nonionic amphiphilic lipids can be present in the nanoemulsionaccording to the invention at a content ranging from 0.2% to 12% byweight, relative to the total weight of the composition, and preferablyranging from 0.2% to 8% by weight, and preferentially ranging from 0.2%to 6% by weight.

The cationic amphiphilic lipids are selected from the list givenhereinafter.

They are present in the nanoemulsions of the invention, preferably, inconcentrations ranging from 0.01 to 6% by weight relative to the totalweight of the nanoemulsion, and more particularly from 0.2 to 4% byweight.

Oils:

The oily phase of the nanoemulsions according to the invention compriseat least one oil. The oils which can be used in the nanoemulsions of theinvention are preferably selected from the group consisting of:

oils of animal or plant origin, made up of esters of fatty acids and ofpolyols, in particular liquid triglycerides, for example sunflower oil,corn oil, soybean oil, avocado oil, jojoba oil, marrow oil, grapeseedoil, sesame seed oil, hazelnut oil, fish oils and glyceryltricaprocaprylate, or plant or animal oils of formula R₉COOR₁₀ in whichR₉ is a higher fatty acid residue having from 7 to 29 carbon atoms andR₁₀ is a linear or branched hydrocarbon-based chain having from 3 to 30carbon atoms, in particular alkyl or alkenyl, for example purcellin oilor liquid jojoba wax;

natural or synthetic essential oils, such as, for example, eucalyptusoil, lavandin oil, lavender oil, vetiver oil, litsea cubeba oil, lemonoil, sandlewood oil, rosemary oil, camomile oil, savoury oil, nutmegoil, cinnamon oil, hyssop oil, caraway oil, orange oil, geraniol oil,cade oil and bergamot oil;

synthetic oils such as parleam oil, polyolefins and liquid carboxylicacid esters;

mineral oils such as hexadecane, isohexadecane and paraffin oil;

halogenated oils, in particular fluorocarbons such as fluoroamines, forexample perfluorotributylamine, fluorohydrocarbons, for exampleperfluorodecahydronaphthalene, fluoro esters and fluoro ethers;

volatile or non-volatile silicone oils.

The polyolefins which can be used as synthetic oils are in particularpoly-α-olefins, and more particularly those of hydrogenated ornon-hydrogenated polybutene type, and preferably hydrogenated ornon-hydrogenated polyisobutene type.

The liquid carboxylic acid esters which can be used as synthetic oilsmay be monocarboxylic, dicarboxylic, tricarboxylic, or tetracarboxylicacid esters. The total number of carbons in the esters is generallygreater than or equal to 10, and preferably less than 100, and moreparticularly less than 80. They are in particular monoesters ofsaturated or unsaturated, linear or branched C₁-C₂₆ aliphatic acids andof saturated or unsaturated, linear or branched C₁-C₂₆ aliphaticalcohols, the total number of carbons in the esters generally beinggreater than or equal to 10. Use may also be made of esters of C₄-C₂₂dicarboxylic or tricarboxylic acids and of C₁-C₂₂ alcohols and esters ofmonocarboxylic, dicarboxylic or tricarboxylic acids and of C₂-C₂₆dihydroxy, trihydroxy, tetrahydroxy or pentahydroxy alcohols.

Among the esters mentioned above, use is preferably made of alkylpalmitates such as ethyl palmitate, isopropyl palmitate, 2-ethylhexylpalmitate, 2-octyldecyl palmitate; alkyl myristates such as isopropylmyristate, butyl myristate, cetyl myristate, 2-octyldodecyl myristate;alkyl stearates such as hexyl stearate, butyl stearate, isobutylstearate; alkyl malates such as dioctyl malate; alkyl laurates such ashexyl laurate and 2-hexyldecyl laurate; isononyl isononanoate; cetyloctanoate.

Advantageously, the nanoemulsions according to the invention contain atleast one oil of molecular weight greater than or equal to 400, inparticular ranging from 400 to 10 000, better still ranging from 400 to5000, or even ranging from 400 to 2500. The oils of molecular weightgreater than or equal to 400 can be selected from oils of animal orplant origin, mineral oils, synthetic oils and silicone oils, andmixtures thereof. As oils of this type, mention may, for example, bemade of isocetyl palmitate, isocetyl stearate, avocado oil and jojobaoil.

The nanoemulsions in accordance with the invention comprise an amount ofoily phase (oil and other fatty substances besides the amphiphiliclipid) preferably ranging from 2 to 40% by weight relative to the totalweight of the nanoemulsion, and more particularly from 4 to 30% byweight, and preferably from 4 to 20% by weight.

The oily phase and the amphiphilic lipids (nonionic and ionicamphiphilic lipids) are preferably present in the nanoemulsionsaccording to the invention according to a weight ratio of the amount ofoily phase to the amount of amphiphilic lipid ranging from 3 to 10, andpreferably ranging from 3 to 6. The term “amount of oily phase” meansthe total amount of the constituents of this oily phase withoutincluding the amount of amphiphilic lipid. The nanoemulsions inaccordance with the present invention can contain, in addition to theurea derivatives of formula (I) described above, solvents, in particularfor improving, if necessary, the transparency of the composition.

These solvents are preferably selected from the group consisting of:

C₁-C₈ lower alcohols such as ethanol;

glycols such as glycerol, propylene glycol, 1,3-butylene glycol,dipropylene glycol, or polyethylene glycols having from 4 to 16 ethyleneoxide units, and preferably from 8 to 12;

sugars such as glucose, fructose, maltose, lactose or sucrose.

These solvents can be used as a mixture. When they are present in thenanoemulsions of the invention, they can be used at concentrationspreferably ranging from 0.01 to 30% by weight relative to the totalweight of the nanoemulsion, and better still from 5 to 20% by weightrelative to the total weight of the nanoemulsion. The amount ofalcohol(s) and/or of sugar(s) preferably ranges from 5 to 20% by weightrelative to the total weight of the nanoemulsion and the amount ofglycol(s) preferably ranges from 5 to 15% by weight relative to thetotal weight of the nanoemulsion.

Method of Preparation:

The method of preparing a nanoemulsion as defined above entails mixingthe aqueous phase containing the urea derivative and the oily phase,with vigorous stirring, at a temperature ranging from 10° C. to 80° C.,and in carrying out a high-pressure homogenization step at a pressure ofgreater than 5×10⁷ Pa and in optionally adding the polymer used.According to a preferred embodiment of the invention, anotherhigh-pressure homogenization step is subsequently carried out at apressure of greater than 5×10⁷ Pa. The high-pressure homogenization ispreferably carried out at a pressure ranging from 6×10⁷ Pa to 18×10⁷ Pa.The shear preferably ranges from 2×10⁶ s⁻¹ to 5×10⁸ s⁻¹, and betterstill from 1×10⁸ s⁻¹ to 3×10⁸ s⁻¹ (s⁻¹ signifies second⁻¹). Such amethod makes it possible to produce nanoemulsions compatible withthermosensitive active compounds, which may contain oils and inparticular fragrances which contain fatty substances, without denaturingthem.

Nanocapsules:

The nanocapsules relating to the invention are those described inEP-0-447,318, EP-0-557,489, EP-0-780,115, EP-1-025,901, EP-1-029,587,EP-1-034,839, EP-1-414,390, FR-2,830,776, EP-1-342,471, FR-2,848,879 andFR 04/50057.

The nanocapsules are Core-Shell particles having an oily core and apolymeric shell. The various applications mentioned above relate tovarious families of polymers and various methods for obtaining them. Thesize of the capsules is always less than 1 μm and it is possible to havesizes of less than 80 nm. These particles can be coated with a lamellarliquid crystal phase most commonly consisting of a lecithin or of adimethicone copolyol. The coating must be an amphiphilic lipid capableof spontaneously forming a lamellar liquid crystal phase on contact withwater. It is to this amphiphilic lipid capable of forming a lamellarphase that the cationic surfactant which will confer on the particles(the nanocapsule) a positive zeta potential will be added. The weightratio of the amphiphilic lipid forming the lamellar phase to thecationic surfactant will be from 99/1 and 75/25. The cationicsurfactants which can be used are those listed hereinafter.

Organic Particles:

The organic particles of the invention are solid nanospheres, which donot have an internal cavity, formed by various methods (dispersion inwater, nanoprecipitation, microemulsion, etc.) and composed of at leastone polymer or of at least one copolymer, or of a mixture thereof. Theparticle is cationic, with the zeta potential defined above, eitherbecause the polymer(s) or copolymer(s) is (are) cationic, or because it(they) is (are) nonionic and a cationic surfactant as describedhereinafter is used. In relation to the polymer, the amount of cationicsurfactant will be from 0 and 25%.

Inorganic Particles:

The cationic inorganic particles of the invention may, by way ofexample, be based on silica, TiO₂, ZnO, alumina, etc. By way of example,representative are alumina particles in a colloidal dispersion in water,such as the Nanomer 2 particles from Nalco. Clariant and Grace alsoprovide particles of this type.

Cationic Surfactants which can be Used for the Preparation of theCationic Particles of the Invention:

The cationic surfactants which can be used according to the inventionare listed hereinafter, this list being non-limiting.

The cationic amphiphilic lipids are preferably selected from the groupconsisting of quaternary ammonium salts and fatty amines and theirsalts.

The quaternary ammonium salts are, for example:

those which have the following general formula (IV):

in which the radicals R₁ to R₄, which may be identical or different,represent a linear or branched aliphatic radical having from 1 to 30carbon atoms, or an aromatic radical such as aryl or alkylaryl. Thealiphatic radicals can contain heteroatoms such as, in particular,oxygen, nitrogen, sulfur or halogens. The aliphatic radicals are, forexample, selected from among the following radicals: alkyl, alkoxy,polyoxy(C₂-C₆)alkylene, alkylamide, (C₁₂-C₂₂)alkylamido(C₂-C₆)alkyl,(C₁₂-C₂₂)alkyl acetate, hydroxyalkyl, containing approximately from 1 to30 carbon atoms; X is an anion selected from the group consisting ofhalides, phosphates, acetates, lactates, (C₂-C₆)alkyl sulfates, andalkyl- or alkylarylsulfonates,

quaternary ammonium salts of imidazolinium, such as, for example, thathaving the following formula (V):

in which R₅ is an alkenyl or alkyl radical having from 8 to 30 carbonatoms, for example derived from tallow fatty acids, R₆ is a hydrogenatom, a C₁-C₄ alkyl radical or an alkenyl or alkyl radical having from 8to 30 carbon atoms, R₇ is a C₁-C₄ alkyl radical, R₈ is a hydrogen atomor a C₁-C₄ alkyl radical, and X is an anion selected from the groupconsisting of halides, phosphates, acetates, lactates, alkyl sulfates,and alkyl- or alkylarylsulfonates. Preferably, R₅ and R₆ denote amixture of alkenyl or alkyl radicals having from 12 to 21 carbon atoms,for example derived from tallow fatty acids, R₇ denotes methyl, and R₈denotes hydrogen. Such a product is, for example, marketed under thetrademark “REWOQUAT W 75” by REWO,

quaternary diammonium salts having the formula (VI):

in which R₉ denotes an aliphatic radical containing approximately from16 to 30 carbon atoms, R₁₀, R₁₁, R₁₂, R₁₃ and R₁₄, which may beidentical or different, are selected from hydrogen or an alkyl radicalhaving from 1 to 4 carbon atoms, and X is an anion selected from thegroup consisting of halides, acetates, phosphates, nitrates and methylsulfates. Such quaternary diammonium salts comprise in particularpropanetallowediammonium dichloride;

quaternary ammonium salts containing at least one ester function.

The quaternary ammonium salts containing at least one ester functionwhich can be used according to the invention are, for example, thosehaving the following formula (VII):

in which:

R₁₅ is selected from among C₁-C₆ alkyl radicals and C₁-C₆ hydroxyalkylor dihydroxyalkyl radicals;

R₁₆ is selected from among:

the radical

linear or branched, saturated or unsaturated C₁-C₂₂ hydrocarbon-basedradicals R₂₀,

a hydrogen atom,

R₁₈ is selected from among:

the radical

linear or branched, saturated or unsaturated C₁-C₆ hydrocarbon-basedradicals R₂₂,

a hydrogen atom,

R₁₇, R₁₉ and R₂₁, which may be identical or different, are selected fromamong linear or branched, saturated or unsaturated C₇-C₂₁hydrocarbon-based radicals;

n, p and r, which may be identical or different, are integers rangingfrom 2 to 6;

y is an integer ranging from 1 to 10;

x and z, which may be identical or different, are integers ranging from0 to 10;

X⁻ is a simple or complex organic or inorganic anion; with the provisothat the sum x+y+z is from 1 to 15, that when x is 0, then R₁₆ denotesR₂₀, and that when z is 0, then R₁₈ denotes R₂₂.

The alkyl radicals R₁₅ may be linear or branched, and more particularlylinear.

Preferably, R₁₅ denotes a methyl, ethyl, hydroxyethyl or dihydroxypropylradical, and more particularly a methyl or ethyl radical.Advantageously, the sum x+y+z is from 1 to 10.

When R₁₆ is a hydrocarbon-based radical R₂₀, it may be long and maycontain from 12 to 22 carbon atoms, or may be short and may contain from1 to 3 carbon atoms.

When R₁₈ is hydrocarbon-based radical R₂₂, it preferably contains 1 to 3carbon atoms.

Advantageously, R₁₇, R₁₉ and R₂₁, which may identical or different, areselected from among linear or branched, saturated or unsaturated C11-C₂₁hydrocarbon-based radicals, and more particularly from linear orbranched, saturated or unsaturated C₁₁-C₂₁ alkyl or alkenyl radicals.

Preferably, x and z, which may be identical or different, are 0 or 1.

Advantageously, y is equal to 1.

Preferably, n, p and r, which may be identical or different, are 2 or 3,and even more particularly are equal to 2.

The anion is preferably a halide (chloride, bromide or iodide) or analkyl sulfate, more particularly methyl sulfate. Use may, however, bemade of methanesulfonate, phosphate, nitrate, tosylate, an anion derivedfrom an organic acid such as acetate or lactate, or any other anioncompatible with the ammonium containing an ester function.

The anion X⁻ is even more particularly chloride or methyl sulfate.

Use is more particularly made of the ammonium salts having the formula(VII) in which:

R₁₅ denotes a methyl or ethyl radical,

x and y are equal to 1;

z is equal to 0 or 1;

n, p and r are equal to 2;

R₁₆ is selected from among:

the radical

methyl, ethyl or C₁₄-C₂₂ hydrocarbon-based radicals;

a hydrogen atom;

R₁₈ is selected from among:

the radical

a hydrogen atom;

R₁₇, R₁₉ and R₂₁, which may be identical or different, are selected fromamong linear or branched, saturated or unsaturated C₁₃-C₁₇hydrocarbon-based radicals, and preferably from linear or branched,saturated or unsaturated C₁₃-C₁₇ alkyl or alkenyl radicals.

Advantageously, the hydrocarbon-based radicals are linear.

Mention may, for example, be made of the compounds having the formula(VII) such as diacyloxyethyldimethylammonium,diacyloxyethylhydroxyethylmethylammonium,monoacyloxyethyldihydroxyethylmethylammonium,triacyloxyethylmethylammonium andmonoacyloxyethylhydroxyethyldimethylammonium salts (chloride or methylsulfate, in particular), and mixtures thereof. The acyl radicalspreferably contain 14 to 18 carbon atoms, and originate moreparticularly from a plant oil such as palm oil or sunflower oil.

When the compound contains several acyl radicals, the latter may beidentical or different.

These products are obtained, for example, by direct esterification oftriethanolamine, of triisopropanolamine, of alkyldiethanolamine or ofalkyldiisopropanolamine, optionally oxyalkylenated, on fatty acids or onmixtures of fatty acids of plant or animal origin, or bytransesterification of their methyl esters. This esterification isfollowed by a quaternization using an alkylating agent such as an alkylhalide (preferably, methyl or ethyl halide), a dialkyl sulfate(preferably, methyl or ethyl sulfate), methyl methanesulfonate, methylpara-toluenesulfonate, glycol chlorohydrin or glycerol chlorohydrin.

Such compounds are, for example, marketed under the trademarks DEHYQUARTby HENKEL, STEPANQUAT by STEPAN, NOXAMIUM by CECA and REWOQUAT WE 18 byREWOWITCO.

The compositions according to the invention preferably contain a mixtureof quaternary ammonium monoester, diester and triester salts with amajority, by weight, being diester salts.

As a mixture of ammonium salts, use may, for example, be made of themixture containing 15 to 30% by weight ofacyloxyethyldihydroxyethylmethylammonium methyl sulfate, 45 to 60% ofdiacyloxyethylhydroxyethylmethylammonium methyl sulfate and 15 to 30% oftriacyloxyethylmethylammonium methyl sulfate, the acyl radicals havingfrom 14 to 18 carbon atoms and originating from optionally partiallyhydrogenated palm oil. Use may also be made of the ammonium saltscontaining at least one ester function described in U.S. Pat. Nos.4,874,554 and 4,137,180.

Among the quaternary ammonium salts having the formula (IV), preferenceis given, firstly, to tetraalkylammonium chlorides, for instancedialkyldimethylammonium or alkyltrimethylammonium chlorides, in whichthe alkyl radical contains approximately from 12 to 22 carbon atoms, inparticular behenyltrimethylammonium, distearyldimethylammonium,cetyltrimethylammonium or benzyldimethylstearylammonium chlorides, orelse, secondly, the stearamidopropyldimethyl (myristyl acetate) ammoniumchloride marketed under the trademark “CERAPHYL 70” by VAN DYK.

According to the invention, behenyltrimethylammonium chloride orbehenyltrimethylammonium bromide and CTAB (cetyltrimethylammoniumbromine) are the quaternary ammonium salts most particularly preferred.

The fatty amines of the invention correspond to the general formula:

wherein:

R₁ is a branched or unbranched, saturated or unsaturatedhydrocarbon-based chain containing from 8 and 30 carbon atoms, andpreferably from 10 and 24;

R₂ and R₃ are selected independently from branched or unbranched,saturated or unsaturated hydrocarbons containing from 1 to 10 carbonatoms, and preferably from 1 to 4;

R₂ and R₃ can also, still independently of one another, correspond to ahydrogen atom H;

M is from 1 to 10, and preferably from 1 to 5.

By way of non-limiting examples, mention will be made of: stearylamine,stearate aminoethylethanolamide, stearyl diethanolamide, stearatediethylenetriamine, stearamidopropyldimethylamine,stearamidopropyldiethylamine, stearamidoethyldiethylamine,stearamidoethyldimethylamine, palmitoamidopropyldimethylamine,palmitoamidopropyldiethylamine, palmitoamidoethyldiethylamine,palmitoamidoethyldimethylamine, behenamidopropyldimethylamine,behenamidopropyldiethylamine, behenamidoethyldiethylamine,behenamidoethyldimethylamine, arachidamidopropyldimethylamine,arachidamidopropyldiethylamine, arachidamidoethyldiethylamine andarachidamidoethyldimethylamine.

As a fatty amine which is commercially available, mention may be made ofIncromine BB from Croda, Amidoamine MSP from Nikkol, and Lexamine fromInolex.

As other fatty amines, mention will, by way of example, be made ofstearylamine, stearate aminoethylethanolamide, stearyl diethanolamideand stearate diethylenetriamine that, inter alia, Sabo sells with theSabomina series.

Mention will also be made of fatty amine acetates such as the Acetamineseries from Kao Corp.

These fatty amines can also be ethoxylated, such as Berol 380, 390, 453and 455, the Ethomeens from Akzo Nobel or Marlazin L10, OL2, OL20,T15/2, T50 from Condea Chemie.

The particles of the present invention can be introduced into anypharmaceutical carrier for cosmetic, dermatological or ophthalmicpurposes. By way of example, mention will be made of lotions, sera, gelsand emulsions of all types.

The compositions according to the invention can be in any of thepharmaceutical forms normally used for topical application, for examplein the form of solutions, gels, dispersions of the lotion or serum type,emulsions which have a liquid or semi-liquid consistency of the milktype, obtained by dispersion of a fatty phase in an aqueous phase (O/W)or conversely (W/O), or suspensions or emulsions which have a soft,semi-solid or solid consistency of the cream or gel type, or elsemicroemulsions, microcapsules, microparticles or vesicular dispersionsof ionic and/or nonionic type. These compositions are prepared accordingto the usual methods.

The compositions according to the invention can also comprise anyadditive normally used in the cosmetics or pharmaceutical field.

Of course, those skilled in the art will take care to select this orthese optional additional compound(s), and/or the amount thereof, insuch manner that the advantageous properties of the compositionaccording to the invention are not, or are not substantially, impaired.

In particular, care will be taken to ensure that this introduction doesnot harm the stability of the cationic particles associated with thesiRNA.

The compositions according to the invention may in particular containcosmetic or pharmaceutical active agents. This will preferably involvedepigmenting agents, sunscreens.

The depigmenting agents which can be incorporated into the compositioncomprise, for example, the following compounds: kojic acid; ellagicacid; arbutin and derivatives thereof such as those described inEP-895,779 and EP-524,109; hydroquinone; aminophenol derivatives such asthose described in WO 99/10318 and WO 99/32077, and in particularN-cholesteryloxycarbonyl-para-aminophenol andN-ethyloxycarbonyl-para-aminophenol; iminophenol derivatives, inparticular those described in WO 99/22707;L-2-oxothiazolidine-4-carboxylic acid or procysteine, and its salts andesters; ascorbic acid and its derivatives, in particular ascorbylglucoside; and plant extracts, in particular extracts of liquorice,mulberry and scullcap, without this list being limiting.

The ultraviolet-radiation-screening agents can be selected from organicUV-screening agents or inorganic UV-radiation-screening agents.

The organic UV-screening agents in accordance with the invention may bewater-soluble, liposoluble or insoluble in the usual cosmetic solvents.They are selected in particular from anthranilates; cinnamicderivatives; dibenzoylmethane derivatives; salicylic derivatives,camphor derivatives; triazine derivatives such as those described inU.S. Pat. No. 4,367,390, EP-863,145, EP-517,104, EP-570,838, EP-796,851,EP-775,698, EP-878,469 and EP-933,376; benzophenone derivatives, inparticular those described in EP-1-046,391 and DE-10012408;β,β′-diphenyl acrylate derivatives, benzotriazole derivatives,benzimidazole derivatives; imadazolines; bisbenzoazolyl derivatives suchas those described in EP-669,323 and U.S. Pat. No. 2,463,264;p-aminobenzoic acid (PABA) derivatives;methylenebis(hydroxyphenylbenzotriazole) derivatives as described inU.S. Pat. Nos. 5,237,071, 5,166,355, GB 2303549, DE 19726184 andEP-893,119; screening polymers and screening silicones such as thosedescribed in particular in WO 93/04665; α-alkylstyrene-derived dimerssuch as those described in DE 19855649; and 4,4-diarylbutadienederivatives such as those described in EP-0-967,200 and DE 19755649.

The inorganic screening agents are generally pigments or elsenanopigments (average size of the primary particles: generally from 5 nmand 100 nm, preferably from 10 nm and 50 nm) of metal oxides which mayor may not be coated, such as, for example, nanopigments of titaniumoxide (amorphous or crystalline in rutile and/or anatase form), of ironoxide, of zinc oxide, of zirconium oxide or of cerium oxide, which areall UV photoprotective agents well known per se. Conventional coatingagents are, moreover, alumina and/or aluminium stearate. Such coated oruncoated metal oxide nanopigments are in particular described inEP-0-518,772 and EP-0-518,773.

The radiation-screening agents in accordance with the invention aregenerally present in the compositions according to the invention inproportions ranging from 0.1 to 20% by weight relative to the totalweight of the composition, and preferably ranging from 0.2 to 15% byweight relative to the total weight of the composition.

The present invention also features the administration of at least onedouble-stranded RNA oligonucleotide of a sequence selected from thegroup consisting of SEQ ID NOS. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 and48 for inhibiting tyrosinase expression.

In particular, the oligonucleotides according to the invention areuseful as a bleaching agent and/or as an anti-browning agent for theskin.

The double-stranded RNA oligonucleotides according to the invention canalso be used for the formulation of topical, cosmetic or dermatologicalcompositions suited to decrease melanin synthesis, in particular with aview to treating hyperpigmentation, and pigmentation marks anddyschromia, or for bleaching hair follicles, regional hyperpigmentationsdue to melanocyte hyperactivity, such as idiopathic melasmas, occurringduring pregnancy (“pregnancy mask” or chloasma) or oestro-progestincontraception, localized hyperpigmentations due to benign melanocytehyperactivity and proliferation, such as senile pigmentation marksreferred to as actinic lentigo, accidental hyperpigmentations, possiblydue to post-lesional wound healing or photosensitization, and alsocertain forms of leukoderma, such as vitiligo. For the latter (the woundhealing possibly resulting in a scar that gives the skin a whiteappearance), since it is not possible to repigment the lesioned skin,the process is completed by depigmenting the areas of residual normalskin so as to impart to the skin as a whole a homogeneous white tint.

The present invention also features a cosmetic method (regime orregimen) for bleaching and/or lightening the complexion and/or makingthe color of a browned skin uniform, comprising the topical applicationof at least one double-stranded RNA oligonucleotide of a sequenceselected from the group consisting of SEQ ID NOS. 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47 and 48.

Preferably, the oligonucleotide is formulated in a topical compositionat a concentration of less than or equal to 1 nM.

More preferably, the oligonucleotide is associated with a cationicparticle less than or equal to 1 μm in size, with a zeta potential offrom 10 to 80 mV, selected from among surfactant micelles, block polymermicelles, liposomes of nonionic and cationic surfactants, niosomes,oleosomes, particles of nanoemulsions, nanocapsules, organic particlesor inorganic particles, as described above.

For the purposes of cosmetic, dermatological, ophthalmic orpharmaceutical care, it is sought to avoid invasive methods such assubcutaneous or ocular injections. The cationic particles associatedwith siRNA according to the present invention can be applied topicallyto the skin or the mucous membranes or in the eye, in suspension orincorporated into an acceptable cosmocentric carrier, such as lotions,sera, emulsified or non-emulsified gels, oil/water, water/oil ormultiple emulsions, microemulsions, etc.

To promote the penetration of the particles associated with siRNA, thewater permeability may be modified; thus, it is possible to control thewater permeability by measuring the IWL (insensible water loss) with aTewameter TM210 or a Dermalab—Cortex technology.

By way of example, the following methods may be used:

stripping (corneodisc, “varnish”), chemical peel or mechanicaldermabrasion before application of the compositions according to theinvention;

pretreatment with a mixture of one or more solvents having a defattingeffect;

cleansing the skin with a detergent foaming product;

occlusive pre- and/or post-treatment either, for example, by coveringthe surface of the skin to be treated with a watertight syntheticmembrane (Blenderm, for example), or by applying a layer of petroleumjelly. This has the effect of blocking the natural IWL of the skin andcausing overhydration of the epidermal lipids which thus become morepermeable.

After application of the compositions of the present invention, methodsmay be employed known to those skilled in the art which promote thepenetration of molecules into the skin, such as, for example,iontophoresis or electroporation.

In the case of an in vitro skin model, the following treatments may becarried out:

before deposition of the composition according to the invention, theCa²⁺ content may be reduced either by changing the medium (0.10-0.75 mMinstead of 1.5 mM), or by adding a chelating agent. This has the effectof decreasing intermembrane adhesion, thus increasing the permeabilityof the skin (Effects of extra- and intracellular calcium concentrationon DNA replication, lateral growth, and differentiation of humanepidermal cells in culture. Virchows Arch B Cell Pathol Incl Mol Pathol.1990; 59(1): 17-25);

using iontophoresis, electroporation and any known methods for modifyingthe permeability of the model.

In order to further illustrate the present invention and the advantagesthereof, the following specific examples are given, it being understoodthat same are intended only as illustrative and in nowise limitative. Insaid examples to follow, all parts and percentages are given by weight,unless otherwise indicated.

EXAMPLE 1 Formulations

Cationic Micelles:

Exemplar 1: Octyl-β-glucoside/cetyltriammonium bromide at the molarratio of 5/1

E1: 100 mM in distilled waterE2: 50 mM in distilled waterE3: 25 mM in distilled waterE4: 12.5 mM in distilled water.

The 2 surfactants (nonionic+cationic) are solubilized in distilledwater. A suspension of the siRNA of SEQ ID NO. 1 (20 μM) is added atfrom 1:1 and 1:9 volume (siRNA: micelles), thus reducing the micellarconcentration in proportion.

Exemplar 2: Micelles of decyl-β-glucoside/behenyltriammonium chloride atthe molar ratio of 5/1 in distilled water. The same concentrations as inthe preceding exemplar are prepared.

These 2 suspensions can be applied to the skin for the treatment ofpigmentation marks and dyschromia or for the bleaching of hairfollicles.

Cationic Liposomes:

Exemplar 1: “Fluid” Vesicles

PEG 400 isostearate 5.5% Behenyltriammonium chloride 0.5% Distilledwater qs 100

Exemplar 2: “Rigid” Vesicles

Sorbitan palmitate 2.75% Cholesterol 2.75% Behenyltriammonium chloride 0.5% Distilled water qs 100

These examples of liposome suspensions are realized by dialysis. Thelipids constituting the vesicles are solubilized in an aqueous solutionof octyl-β-glucoside. This solution is then dialyzed against water for72 h.

The suspension of siRNA SEQ ID NO. 4 is then added, bringing theconcentration with respect to the vesicle to around 3% of lipid. Thissuspension of nonionic liposomes can be applied to the skin for thetreatment of pigmentation marks and dyschromia or for the bleaching ofhair follicles.

Cationic Oleosomes:

Oil Phase:

Sucrose mono-distearate marketed by 0.45% Stéaraineries Dubois Sorbitanstearate 4EO (Tween 61 Uniquema) 0.30% Behenyltriammonium chloride 0.21%Vit E acetate   0.5%: Jojoba oil  0.5% Stearyl heptanoate   1% Volatilesilicone oil   1% Vit F glyceride  0.5% Preservative 0.02% BHT 0.01%

Aqueous Phase:

Distilled water qs 100 Preservative 0.1%

This dispersion is obtained by high-pressure homogenization in order tohave a particle size of approximately 170 nm. A suspension of siRNA SEQID NO. 16 (20 μM) at ratios ranging from 1:1 to 1:20 (siRNA/oleosomes)is then added. The siRNA will then complex with the surface of theparticles.

Cationic Nanocapsules:

Organic Phase:

Polycaprolactone (MW: 50 000) 1% Vit E 1% Dimethicone copolyol DC2 5695(Dow Corning) 0.5%   Behenyltriammonium chloride 0.21%   Acetone 200 ml

Aqueous Phase:

Pluronic F68 0.5% Distilled water 200 ml

The organic phase is introduced with stirring into the aqueous phase.The acetone and 100 ml of aqueous phase are then evaporated off so as toobtain the suspension of nanocapsules, the size of which is 220 nm. Asuspension of siRNA SEQ ID NO. 37 (20 μM) at ratios ranging from 1:1 to1:20 (siRNA/nanocapsules) is then added. The siRNA will then complexwith the surface of the particles.

Organic Nanoparticles:

Organic Phase:

Polyethylene adipate   2% (Scientific Polymer products) Dimethiconecopolyol DC2 5695 (Dow Corning)  0.5% Behenyltriammonium chloride 0.21%Acetone 200 ml

Aqueous Phase:

Pluronic F68 0.5% Distilled water 200 ml

The organic phase is introduced with stirring into the aqueous phase.The acetone and 100 ml of aqueous phase are then evaporated off so as toobtain the suspension of nanoparticles, the size of which is 180 nm. Asuspension of siRNA SEQ ID NO. 40 (20 μM) at ratios ranging from 1:1 to1:20 (siRNA/nanoparticles) is then added. The siRNA will then complexwith the surface of the particles.

Cationic Nanoemulsions:

Oil Phase:

PEG 400 isostearate marketed by Uniqema 1% Behenyltriammonium chloride1% Avocado oil 1% Jojoba oil 3% Cyclopentamethylsiloxane 2%

Aqueous Phase:

Distilled water 30% Dipropylene glycol 10%

Dilution Phase:

Distilled water qs 100% Preservative 0.1%

An emulsion is prepared by dispersing the oily phase in the aqueousphase with very vigorous stirring. The suspension obtained is thenhomogenized several times using a very-high-pressure homogenizer, at apressure of approximately 1200 b. The particle size is of the order of50 nm and the suspension is transparent. The dilution phase is thenadded.

As in the previous cases, a suspension of siRNA SEQ ID NO. 42 (20 μM) atratios ranging from 1:1 to 1:20 (siRNA/nanoemulsion) is then added. ThesiRNA will then complex with the surface of the particles.

EXAMPLE 2 Measurement of the Activity of the siRNAs According to theInvention

The effectiveness of the 47 siRNAs of sequence SEQ ID NOS 1 to 47according to the invention was evaluated in the “pSCREEN-iT™ system”test developed by Invitrogen, set up to evaluate the effectiveness ontyrosinase inhibition (GenBank accession number M27160).

Experimental protocol for the test:

-   -   2×10⁴ A549 cells/well are seeded into a 48-well plate and        cultured overnight.    -   Cotransfection is carried out in triplicate for 5 h using 2        μg/ml of Lipofectamine 2000 (Invitrogen) complexed with:        -   tyrosinase pScreen-iT™ vector coupled to the LacZ gene—10            ng/well        -   luciferase reporter plasmid—20 ng/well        -   1 nM Stealth™ RNA    -   The cells are lysed 24 h after the transfection and the        β-galactosidase (β-Gal) and luciferase (Luc) activities are        measured. The β-Gal/Luc ratio determines the percentage        inhibition of tyrosinase.

The results obtained are reported in Table I.

TABLE I Assaying of the effectiveness of the 47 double-stranded RNAoligonucleotides on the degradation of tyrosinase mRNA (GenBankaccession number M27160). Oligonucleotide % Oligonucleotide % (SEQ IDNO.) Inhibition (SEQ ID NO.) Inhibition 1 97.50% 22 77.91% 2 98.57% 2397.29% 3 86.95% 24 90.50% 4 97.95% 25 95.17% 5 94.17% 26 90.60% 6 77.68%27 87.11% 7 95.86% 28 91.58% 8 97.50% 29 96.00% 9 87.14% 30 90.39% 1091.54% 31 10.38% 11 88.44% 32 97.93% 12 89.50% 33 84.79% 13 98.15% 3464.43% 14 97.87% 35 97.71% 15 36.02% 36 92.12% 16 97.78% 37 97.07% 1788.54% 38 9.55% 18 81.11% 39 90.22% 19 40.78% 40 97.44% 20 83.33% 4188.11% 21 92.64% 42 98.71% 43 99.10% 46 98.64% 44 83.54% 47 94.14% 4589.05%

Secondly, a dose-effect was determined on the 20 sequences which weremost effective, i.e., with an effectiveness of greater than 91% at 1 nM.The results are reported in Table II.

TABLE II Assaying of the effectiveness of the 20 sequences selected, bydose-effect with doses of 0.0016 nM to 1 nM. Oligonucleotide (SEQ IDNO.) 1 nM 0.25 nM 0.063 nM 0.016 nM 1 97.37% 94.34% 89.47% 72.07% 297.98% 95.64% 89.34% 66.68% 4 97.93% 95.22% 90.24% 57.92% 5 91.33%79.88% 61.47% 18.40% 7 96.86% 88.97% 76.45% 38.34% 8 97.84% 90.76%81.12% 38.57% 13 98.13% 95.73% 91.11% 62.56% 14 97.59% 87.22% 68.77%33.87% 16 97.95% 94.92% 83.40% 56.25% 23 96.05% 87.42% 62.32% 29.97% 2593.45% 90.50% 78.36% 46.80% 29 96.42% 88.24% 70.64% 31.15% 32 96.88%90.69% 80.33% 35.04% 35 97.42% 94.84% 88.34% 54.29% 37 96.38% 92.46%88.24% 53.92% 40 97.65% 95.08% 91.59% 60.64% 42 98.25% 96.54% 92.58%53.46% 43 98.46% 95.60% 89.21% 38.85% 46 98.77% 94.81% 84.23% 48.74% 4794.60% 76.71% 52.94% 30.18%

The siRNAs according to the present invention show an effectiveness ofgreater than 94% at 1 nM for the 20 sequences most effective. The lowestdose tested is 0.016 nM for an effectiveness of 72% on degradation ofthe mRNA encoding tyrosinase.

EXAMPLE 3 Evaluation of a Possible Interferon-Type Response Caused bythe siRNAs According to the Invention

It was verified that the siRNAs which are subjects of the presentinvention did not induce an interferon-type response. For this, theexpression of the OAS-1 and IFIT-1 (interferon-inducibletetratricopeptide repeat domain) genes, known to be induced byinterferon (Wieland S F et al., Searching for Interferon-Induced GenesThat Inhibit Hepatitis B Virus Replication in Transgenic MouseHepatocytes, J. of Virology 2003; 77: 1227-1236. Persengiev S P et al.,Nonspecific, concentration-dependent stimulation and repression ofmammalian gene expression by small interfering RNAs (siRNAs). RNA 2004;10: 12-18. Bridge A J et al., Induction of an interferon response byRNAi vectors in mammalian cells. Nature Genet. 2003; 34: 263-264.Scacheri P C et al., Short interfering RNAs can induce unexpected anddivergent changes in the levels of untargeted proteins in mammaliancells. Proc. Natl. Acad. Sci. USA 2004, 101: 1892-1897. Sledz C A etal., Activation of the interferon system by short-interfering RNAs. NatCell Biol. 2003; 9: 834-9. Patzwahl R et al., Enhanced expression ofinterferon-regulated genes in the liver of patients with chronichepatitis C virus infection: detection by suppression-subtractivehybridization. J. Virol. 2001; 75: 1332-1338.), was measured byquantitative PCR (qPCR) according to the protocol described in the“BLOCK-iT™ RNAi Stress Response Control Kit (Human) for monitoringinterferon-mediated stress response to double-stranded RNA in humancells” marketed by Invitrogen.

Measurement of the Level of Expression of the hOAS-1 Gene:

The level of expression of the hOAS-1 gene was related to that of theGAPDH reference gene.

hOAS-1/ Sample GAPDH Stdev dsRNA of SEQ ID NO. 1 - 1 nM 0.608 0.046dsRNA of SEQ ID NO. 2 - 1 nM 0.668 0.292 dsRNA of SEQ ID NO. 4 - 1 nM0.608 0.070 dsRNA of SEQ ID NO. 13 - 1 nM 1.232 0.866 dsRNA of SEQ IDNO. 46 - 1 nM 0.911 0.221 dsRNA of SEQ ID NO. 1 - 50 nM 1.080 0.500Control sequence at 1 nM 2.141 1.336 Positive control (long dsRNA of 1kb) 24.905 6.431 dsRNA of SEQ ID NO. 1 - 0.1 nM 0.856 0.534 dsRNA of SEQID NO. 2 - 0.1 nM 0.866 0.300 dsRNA of SEQ ID NO. 4 - 0.1 nM 0.966 0.568dsRNA of SEQ ID NO. 13 - 0.1 nM 1.238 0.451 dsRNA of SEQ ID NO. 46 - 0.1nM 1.262 0.328 Control sequenceat 50 nM 0.807 0.404 Control sequence at0.1 nM 1.949 1.502

Result: Only the positive control (long dsRNA of 1 kb) induces asignificant increase in hOAS-1 gene expression. The sequences tested(SEQ ID NOS. 1, 2, 4, 13 and 46) do not significantly increase hOAS-1gene expression.

Measurement of the Level of Expression of the hIFIT-1 Gene:

Threshold Sample cycle Stdev dsRNA of SEQ ID NO. 1 - 1 nM 36.15 0.20dsRNA of SEQ ID NO. 2 - 1 nM 32.77 1.85 dsRNA of SEQ ID NO. 4 - 1 nM35.98 0.88 dsRNA of SEQ ID NO. 13 - 1 nM 35.41 1.88 dsRNA of SEQ ID NO.46 - 1 nM 34.42 3.24 dsRNA of SEQ ID NO. 1 - 50 nM 34.16 3.02 Controlsequence at 1 nM 31.30 1.98 Positive control (long dsRNA of 1 kb) 25.700.79 dsRNA of SEQ ID NO. 1 - 0.1 nM 32.47 3.07 dsRNA of SEQ ID NO. 2 -0.1 nM 35.38 1.41 dsRNA of SEQ ID NO. 4 - 0.1 nM 34.86 1.86 dsRNA of SEQID NO. 13 - 0.1 nM 33.22 2.33 dsRNA of SEQ ID NO. 46 - 0.1 nM 33.89 2.03Control sequence at 50 nM 35.70 3.30 Control sequence at 0.1 nM 33.653.90

Result: Only the positive control (long dsRNA of 1 kb) induces asignificant expression of the hIFIT-1 gene detected at a threshold cyclevalue of approximately 25. The sequences tested (SEQ ID NOS. 1, 2, 4, 13and 46) induce hIFIT-1 gene expression only for threshold cycle valuesof approximately 32-36, i.e., a difference of 7-11 cycles correspondingto an amount of messengers which is 10²-10³ times lower.

These assays make it possible to conclude that the dsRNAs according tothe invention do not induce an interferon-type response.

Each patent, patent application, publication, text and literaturearticle/report cited or indicated herein is hereby expresslyincorporated by reference.

While the invention has been described in terms of various specific andpreferred embodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions, and changes may be made withoutdeparting from the spirit thereof. Accordingly, it is intended that thescope of the present invention be limited solely by the scope of thefollowing claims, including equivalents thereof.

1. A cosmetic/pharmaceutical composition comprising at least onedouble-stranded RNA oligonucleotide of a sequence selected from thegroup consisting of SEQ ID NOS. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 and48 together with a population of cationic particulates less than orequal to 1 μm in size, and having a zeta potential ranging from 10 to 80mV.
 2. The cosmetic/pharmaceutical composition as defined by claim 1,said at least one RNA oligonucleotide being -o-methylated at the2′-position thereof.
 3. The cosmetic/pharmaceutical composition asdefined by claim 1, comprising less than or equal to 100 μM of said atleast one RNA oligonucleotide.
 4. The cosmetic/pharmaceuticalcomposition as defined by claim 1, said population of cationicparticulates comprising surfactant micelles, block polymer micelles,liposomes of nonionic and cationic surfactants, niosomes, oleosomes,particles of nanoemulsions, nanocapsules, organic particles or inorganicparticles.
 5. The cosmetic/pharmaceutical composition as defined byclaim 1, said cationic particulates being less than or equal to 500 nmin size.
 6. The cosmetic/pharmaceutical composition as defined by claim1, said cationic particulates being less than or equal to 300 nm insize.
 7. The cosmetic/pharmaceutical composition as defined by claim 1,said cationic particulates comprising micelles of nonionic amphiphilicsurfactants and of cationic surfactants.
 8. The cosmetic/pharmaceuticalcomposition as defined by claim 4, comprising block polymer micellesselected from the group consisting of micelles of a cationic amphiphilicblock polymer, micelles of a nonionic amphiphilic block polymer and of acationic amphiliphic block polymer and micelles of a nonionicamphiphilic block polymer and of a cationic surfactant.
 9. A regime orregimen for treating idiopathic melasmas, hyperpigmentations associatedwith pregnancy or with oestro-progestin contraception, accidentalhyperpigmentations, hyperpigmentations due to leukoderma, and/orvitiligo, comprising topically applying onto the skin of an individualin need of such treatment, a thus effective amount of at least onedouble-stranded RNA oligonucleotide of a sequence selected from thegroup consisting of SEQ ID NOS. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 and 48together with a population of cationic particulates less than or equalto 1 μm in size, and having a zeta potential ranging from 10 to 80 mV.10. The cosmetic/pharmaceutical composition as defined by claim 1,formulated into a topically applicable medium therefor, said at leastone RNA oligonucleotide comprising less than or equal to 100 μM thereof.11. The cosmetic/pharmaceutical composition as defined by claim 1,formulated as a solution, gel, lotion, serum, milk, emulsion,dispersion, suspension, cream, microcapsules, microparticles, orvesicular dispersion.